Combined Haemophilus influenzae type b and pertussis vaccine

ABSTRACT

A process for the isolation and purification of immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b. The PRP has been purified with ethanol, Cetavlon (hexadecyltrimethyl ammonium bromide) and a phosphate containing adsorbent, hydroxylapatite. The contaminants (nucleic acid, proteins and endotoxins) are removed to the minimum level by a treatment with hydroxylapatite. Also described is a process for the preparation of a combined vaccine containing the PRP (prepared as aforementioned) and B. pertussis antigens. The vaccine elicits anti-PRP antibody and antipertussis antibody (as measured by microagglutination) formations in young animals. This sera with anti-PRP antibody exhibits a strong bactericidal activity.

BACKGROUND OF THE INVENTION

Applicant is not aware of any prior art references which, in hisjudgement as one skilled in the art, would anticipate or render obviousthe instant invention; however, for the purpose of fully developing thebackground of the invention and establishing the scope and content ofthe prior art, the following references are set forth:

(1) L. P. Rodriques, R. Schneerson and J. B. Robbins, Immunity toHaemophilus influenzae type b; I The Isolation and Some Physical,Chemical, Serologic and Biologic Properties of the CapsularPolysaccharide of Haemophilus influenzae type b. Journal of Immunology,107, 1071-1079, (1971). This article describes the removal of endotoxinsand pyrogenic substances from the polyribosyl ribitol phosphate usingchloroform and t-butanol;

(2) P. Anderson and D. H. Smith, Isolation of the CapsularPolysaccharide from the Culture Supernatant of Haemophilus influenzaetype b. Infection and Immunity, 15, 472-477 (1977). This articledescribes a method for removing endotoxins and pyrogenic substances frompolyribosylribitol phosphate using phenol at 4° C. followed by highspeed (100,000×g) centrifugation for several hours;

(3) Robbins, J. B., J. C. Parke., R. Schneerson, and J. K. Whisnant,1973. Quantitative measurement of "Natural" and immunization-inducedHaemophilus influenzae type b capsular polysaccharide antibodies.Pediatr. Res. 7:103-110;

(4) Smith, D. H., G. Peter, D. L. Ingram, A. L. Harding, and P.Anderson. 1973. Children immunized against Haemophilus influenzae typeb. Pediatrics 52:637-644;

(5) Anderson, P., D. H. Smith, D. L. Ingram, J. Wilkins, P. F. Wehrle,and V. M. Howie. 1977. Antibody to polyribophosphate of Haemophilusinfluenzae type by in infants and children: Effect of immunization withpolyribophosphate. J. Infect. Dis., 136 (Suppl.): 557-562. Thisreference teaches that neither purified PRP alone, or in combinationwith Diphtheria, pertussis and tetanus provoke humoral activity in younginfants against H. influenzae type b.

(6) Moxon, E. R., P. Anderson, D. H. Smith, B. Adrianzen, G. G. Graham,and R. S. Baker. 1975. Antibody responses to a combination vaccineagainst Haemophilus influenzae type b, diphtheria, pertussis, andtetanus. Bull. W. H. O. 52:87-90. This reference teaches that thecombined vaccine of PRP and diptheria-pertussis vaccine was lesseffective than PRP alone; and

(7) Limjuco, G. A. and D. J. Carlo. 1976. Endotoxin free Meningococcuspolysaccharides. U.S. Pat. No. 3,978,209.

(1) Field of the invention

This invention is in the field of vaccines for immunization againstHaemophilus influenzae type b infections, such as meningitis. Morespecifically, this invention relate to: (1) a method for the isolationof antigenic polysaccharide polyribosyl ribitol phosphate (PRP) fromHaemophilus influenzae type b cultures; and, (2) a process for thepreparation of a combined vaccine containing PRP and B. pertussisantigens. The PRP of this invention should also be effective when usedin conjunction with other non-pathogenic strains of bacteria, such as E.coli, B. subtilis, S. aureus, B. punilus and L. plantarum, to trigger anantibody response in warm-blooded animals.

(2) Description of the prior art

The purified polyribitol phosphate (PRP) from Haemophilus influenzaetype b is being investigated as a protective immunogen, however, anactive antigenic response in young animals and infants has not beenachieved (see prior art references 1, 3, 4, 5). The prior art method forthe purification employed ethanol and Cetavlon (hexadecyltrimethylammonium bromide). The contaminants, endotoxins, and pyrogenicsubstances were removed by the use of cold phenol or chloroform andt-butanol which may result in the loss of the antigenic nature of thispolysaccharide (see prior art references 1, 2, 7).

SUMMARY OF THE INVENTION

A new process for the isolation and purification of immunologicallyactive PRP from Haemophilus influenzae type b has been established.

The process to be described has distinct advantages over the prior artprocedures in that all contaminants (nucleic acids, proteins andendotoxins) are removed to the minimum level by a treatment withhydroxylapatite. The PRP prepared by the process described here gives ahigher molecular weight polysaccharide than those reported previously(see prior art references 1, 3, 4).

More importantly, the PRP prepared by this new procedure is highlyimmunogenic in warm-blooded animals as opposed to the PRP produced byprior art procedures.

A process for removal of contaminants (proteins, nucleis acids andendotoxins) in the polysaccharide PRP is performed by treating thepartial purified polysaccharide with a phosphate containing adsorbentwhich does not absorb the polysaccharide under designated conditions.

The second objective of this invention is to provide a process for thepreparation of a combined PRP and pertussis vaccine which is highlyimmunogenic in young animals.

(I) ISOLATION AND PURIFICATION OF POLYRIBOSYL RIBITOL PHOSPHATE, THECAPSULAR POLYSACCHARIDE OF HAEMOPHILUS INFLUENZAE TYPE B. Organisms,Growth Medium and Culture

Two strains of Haemophilus influenzae type b are used. The Rab strain isobtained from Grace Leidy, Babies Hospital, Columbia University, NewYork City, N.Y. The CK strain was isolated from a patient at WaterburyHospital Waterbury, Conn. The organisms are passed through mice severaltimes to insure their virulence. The organisms are isolated at autopsyfrom the brain tissue of mice, subcultured on either 3.7% Brain HeartInfusion (BHI) (Difco Lab., Detroit, Mich.) medium or on 5% BHI agarsupplemented with 0.01% nicotinamide adenine dinucleotide (NAD) (P-LBiochemicals, Milwaukee, Wis.) and 1% (v/v) defibrinated horse blood(Animal Blood Center, Syracuse, N. Y.) and then distributed in one mlportions in ampules, lyophilized and stored at -70° C.

Basal medium used for the growth of the organisms is 3.7% (BHI). Thebasal medium is supplemented with 10 mg of NAD and 20 mg of hemin(Eastman Kodak, Rochester, N. Y.) per liter. One percent (v/v)defibrinated horse blood is added per 50 ml of seed culture. Thesupplements are freshly prepared and filtered through a 0.45μ Nalgenefilter unit Nalge Sybron Corp., Rochester, N. Y.) before use. For growthof the organism in a 14 liter fermentor, the medium is furthersupplemented with 0.5% glucose.

To prepare a flask of seed organisms, one ml of frozen stock culture isthawed and transferred to 50 ml of the enriched BHI medium supplementedwith defibrinated horse blood and NAD. The culture is grown for 8 hoursat 37° C. on a gyrotory shaker at 150 rpm. The organisms are added as a1% inoculum to 500 ml portions of the enriched BHI broth in 2 literflasks which are then incubated at 37° C. with moderate shaking on agyrotory shaker for 8 hours (for isolation of PRP) or 14 hours (as seedcultures).

The fermentation of each batch in a 14 liter fermentor is initiated byaseptically transferring 700 ml of the seed culture to 7 liters of theenriched BHI broth supplemented with hemin instead of defibrinated horseblood. The culture is continuously maintained at 37°±1° C. The tank isstirred at a rate of 150 rpm and an air flow of 0.25 liter of air perliter of mash per minute is maintained. During the growth 0.001% of asilicone antifoam (FD-82, Hodag Chemical Corp.) is added as needed.Cultures are grown to the late logarithmic of growth (8 to 10×10⁹ viablecells/ml), usually about 8 hours and then growth is terminated by adding0.4% formaldehyde and standing overnight at 4° C.

Isolation of Polyribosyl Ribitol Phosphate (PRP)

Culture broth prepared as described above is centrifuged at 27,000×g for30 minutes at 4° C. The cell free supernatant is collected and treatedas follows:

Step 1, Ethanol Precipitation

To the culture supernatant is added sodium acetate (final concentration4%). The solution is adjusted to pH 6.0-6.2 and 44 liters of 3A ethanolare added slowly with vigorous stirring at 4° C. The mixture is adjustedto pH 6.8 with glacial acetic acid and then allowed to stand for 12hours at 3° C. The resulting precipitate is collected by decantation andthen centrifugation to afford crude PRP.

Step 2, Cetavlon(hexadecyltrimethyl ammonium bromide)Treatment

The precipitate from Step 1 is dissolved in pyrogen-free distilled waterand centrifuged to remove the residue. The clear brown solution is thenslowly added to 100 ml of a 10% aqueous solution of Cetavlon with mixing(final conc. 0.5%). The mixture is stirred for one hour and thencentrifuged. The precipitate of nucleic acids and PRP-Cetavlon complexis mixed with 2 liters of 0.3 M sodium chloride. The cloudy solution iscentrifuged to eliminate insoluble materials such as nucleic-Cetavlonsalt.

The supernatant is diluted with an equal volume of water causing thePRP-Cetavlon salt to precipitate. The mixture is stirred for one hour,the precipitate is collected by centrifugation and is then dissolved in2 liters of 0.3 M sodium chloride.

Step 3, Ethanol Precipitation

Cetavlon and the contaminants of nucleic acids and proteins are furtherremoved by ethanol precipitation (at least 2 times). The PRP isprecipitated as described in Step 1 while Cetavlon is dissolved in thealcoholic solution. The PRP is recovered by centrifugation, dissolved in2 liters of pyrogen-free distilled water and then reprecipitated asdescribed above. The final PRP precipitate is solubilized in 20 mMsodium phosphate, pH 6.8.

Step 4, Hydroxylapatite Treatment

Contaminants (e.g. nucleic acids, proteins and endotoxins) in thepartial purified PRP preparations are selectively removed by adsorptionon a calcium phosphate containing adsorbent such as hydroxylapatite[3.Ca₃ (PO₄)₂.Ca(OH)₂ ].

This invention is based on the discovery that the polysaccharide PRP isnot adsorbed by the calcium phosphate adsorbent containing phosphatebuffer (20 mM) having a pH of about 6.7-6.9; or a phosphate buffer (50mM) having a pH of about 5.8; however, the contaminants (such as nucleicacids, proteins and endotoxins) are adsorbed under these conditions. Theprocess of the present invention may be carried out in batch-wise orcolumn operation. In batch process, the hydroxylapatite is added to thepartially purified PRP preparation (in 20 mM phosphate; pH 6.9). Themixture is mixed well and centrifuged to remove non-desired solids(adsorbent and the contaminants adsorbed by the adsorbent). Thesupernatant fluid is subjected to the foregoing procedure at least 2more times. The resulting solution is filtered through milliporefilters, dialyzed against pyrogen-free distilled water, and lyophilized.

In column operation, the partially purified PRP in 20 mM phosphatebuffer (pH of at least 5.8) is applied to a column containing theadsorbent hydroxylapatite which had been equilibrated with 20 mMphosphate buffer, pH 5.8 and eluted with a stepwise gradient of sodiumphosphate buffer (pH 5.8) from 20 mM to 100 mM. Fractions are collectedand assayed for pentose (for polyribosyl ribitol phosphate). Thosefractions which are positive for pentose are dialyzed againstpyrogen-free distilled water and lyophilized.

(II) A PROCESS FOR PREPARING A COMBINATION VACCINE CONSISTING OF PRPFROM H. INFLUENZA TYPE b AND B. PERTUSSIS ANTIGENS

To prepare a PRP vaccine, lyophilized PRP (prepared as aforementioned)is dissolved at a concentration of 20 ug/ml in phosphate buffered saline(PBS) (0.113 g potassium diphosphate, 0.83 g disodium phosphate, and 8.5g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal). Thevaccine is sterile filtered through 0.45μ millipore filter units,dispensed into glass vials, and stored at 4° C.

A concentrated solution of the PRP is prepared by weighing predeterminedamounts of the polysaccharide and dissolving it in phosphate bufferedsaline (0.113 g potassium diphosphate, 0.83 g disodium phosphate, and8.5 g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal).This concentrated solution of PRP is then mixed with an appropriatevolume of fresh B. pertussis cell suspensions to prepare the stocksolution. The combined vaccine in this stock solution contains 200 μg ofPRP and approximately 70 opacity units (op) of cells per ml. The stocksolution is kept at 4° C. for 90 days to allow for detoxification ofpertussis antigens before the final product (vaccine) is prepared (whichcontains 10 μg of PRP and 3.5 op units of pertussis cells/0.5 ml dose).

DETAILED DISCLOSURE Example 1

Hydroxylapatite (e.g. Bio. gel HTP, Bio-Rad Laboratories, Richmond,Calif.), 2.5 g is added to 250 ml partially purified PRP preparations(after Cetavlon and ethanol treatments) (containing approximately 1.0 mgPRP/ml) in 20 mM sodium phosphate buffer, pH 6.9 and mixed at ice-coldwater bath (1°-4° C.) for one hour. The mixture is centrifuged in theSorvall RC2-B for 30 minutes at 16,000×g. The supernatant fluid is thenpassed through 0.65μ millipore filter and subjected to the foregoingprocedure (each time treated with 2.5 g hydroxylapatite) 2 more times.The resulting solution is filtered through 0.65μ and 0.45μ milliporefilters, dialyzed against pyrogen-free distilled water and lyophilized.The lyophilized product exhibits strong immunogenic activity (see belowcombined vaccine and animal experiment) and contains very lowcontaminants (such as nucleic acids, proteins and endotoxins) (see TableI below). Approximately 170 mg of the lyophilized PRP is obtained. Therecovery of PRP by this process is approximately 70% of the startingpolysaccharide. The PRP should be stored under suitable conditions, suchas at 4° C. in a desiccator over phosphorus pentoxide and silica gel.

Example 2

A partially purified PRP (300 mg PRP) is dissolved in 100 ml of 20 mMsodium phosphate buffer, pH 5.8 (i.e. 3 mg PRP/ml). This solution isapplied to a column at ambient temperature (5.0×45 cm) ofhydroxylapatite (approximately 250 ml bed volume which has beenequilibrated with 20 mM sodium phosphate buffer, pH 5.8 and eluted witha stepwise gradient of 20 mM, 50 mM, 100 mM sodium phosphate buffer, pH5.8. The individual fractions (200 ml) eluted with 20 mM and 50 mMsodium phosphate buffer (pH 5.8), positive for pentose (pentosedetermination by the orcinal method) are collected, dialyzed againstpyrogen-free distilled water and lyophilized. Approximately 206 mg ofthe lyophilized product, PRP, is obtained.

The purity of the polysaccharide, PRP is assayed by estimating to whatextent they are contaminated with nucleic acids, proteins andendotoxins. Protein concentration is determined by the method of Lowry,et al. in J. Biol. Chem. 193:265 (1951) with boving serum albumin asstandard. Nucleic acid is measured by the absorption of the PRP solutionat 260 nm. The absorbance of 50 μg of nucleic acid in one ml of water ina cell of 1-cm light path is assumed to be equal to 1.0. Molecular sizeis estimated by means of Sepharose 4B or 2B gel filtration on columns1.5×90 cm (Pharmacia-Fine Chemicals, Piscataway, N. J.). Partitioncoefficient (Kd) values are calculated from the elution patterndeveloped by the orcinal reaction (Herbert, et al., Methods inMicrobiology, Vol. 5B, 285-291.

                                      Table I                                     __________________________________________________________________________    Physical-Chemical Characteristics of PRP Prefered                             Assay                       Endotoxin                                               Mol. Size                                                                           Pentose                                                                            Nucleic Acid                                                                         Protein                                                                           Rabbit Pyrogen                                                                        Limulus Lysate                            Procedure                                                                           (Kd)  (%)  (%)    (%) Test.sup.c                                                                            Test.sup.d                                __________________________________________________________________________    Example 1                                                                           0.sup.a                                                                             36.0 0.8    0.7 10.0    1/400                                     Example 2                                                                           0.44.sup.b                                                                          33.8 0.3    0.7 10.0    1/1000                                    __________________________________________________________________________     a) Using both Sepharose 2B and 4B, molecular weight>                          b) Using Sepharose 4B                                                         c) μg PRP/Kg rabbit body weight not yielding fever                         d) Reciprocal dilution of PRP (100 μg PRP/ml) when compared to Bureau      of Biologics E1 (endotoxin standard)                                     

Example 3

A combination vaccine containing PRP from H. influenzae type b and B.pertussis antigens is prepared as follows: 140 mg PRP (as prepared inExample 2) is dissolved in 350 ml sterile phosphate buffered saline(PBS) (0.113 g potassium diphosphate, 0.83 g disodium phosphate, and 8.5g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal), andfilter through 0.45μ millipore filter. This concentrated PRP solution(400 μg/ml) is used to prepare a combination vaccine which consists ofPRP and B. pertussis antigens.

To 150 ml of the concentrated PRP solution (400 μg/ml) add 150 ml offresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0(containing 149 opacity; op, unit cells/ml) to prepare the stocksolution of the combined vaccine. This stock solution (300 ml) is keptat 4° C. until the endotoxin level of the pertussis is decreased (atleast 90 days). The sterility of the stock solution is tested withthioglycollate media (at 32-33° C.). The pH of the stock solution after90 days incubation is checked and adjusted to pH 7.0±1. The finalproduct (vaccine) used for animal experiments is prepared by dilutingthe stock combined solution with PBS and it contains 10 μg of PRP and3.5 op units of pertussis cells/0.5 ml (dose).

The results of the antibody response to this combined vaccine in youngrats appears in FIGS. 1 and 2 as shown.

Example 4

The stock solution of PRP (400 μg/ml) is prepared by dissolving 30 mgPRP (as prepared in Example 1) in 75 ml sterile phosphate bufferedsaline (PBS) and filtered through 0.45μ millipore filter. To 25 ml ofthis concentrated PRP is added an equal volume (i.e. 25 ml) of fresh B.pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 opunits cells/ml) to prepare the stock solution of the combined vaccine.This stock solution (50 ml) is kept at 4° C. for (at least) 90 days. Thesterility of the stock solution is tested as aforementioned withthioglycollate media. The pH of the stock solution is pH 7.0±1. Thefinal product (vaccine) used for animal experiments is prepared bydiluting the stock combined solution with PBS, and it contains 10 μg ofPRP and 3.7 op units of pertussis cells/0.5 ml (dose).

Example 5

The PRP stock solution (200 μg/ml in PBS (pH 7.0) is prepared as inExample 4. The pertussis stock solution (containing 75 op units/ml) isprepared by diluting 25 ml of fresh B. pertussis cell suspension (149 opunits/ml) with an equal volume of PBS. Both stock solutions areincubated separately at 4° C. for 90 days or slightly longer. Thecombined vaccine is made by taking 14 ml of PRP stock solution (200μg/ml), plus 14 ml (detoxified) stock solution of pertussis antigens (75op units/ml) and diluting it with 112 ml PBS (final val. 140 ml). Thefinal vaccine used for animal experiments contains 10 μg PRP and 3.7 opunits pertussis/0.5 ml (dose).

The results of the antibody response to this combined vaccine in youngrats appears in FIG. 3 and in Table II.

                                      TABLE II                                    __________________________________________________________________________    Immunogenicity of PRP-Pertussis Complex in Young Rats .sup.a                                  Anti-PRP Antibody Activity (cpm .sup.3 H-PRP                                  Bound/50μ1 Serum)                                                     Dose                   3rd Week Post-injection                                (single or                                                                         Week Post-Injection                                                                             (fold increase from                         Vaccine    multiple)                                                                          O  1    2    3    preimmunization)                            __________________________________________________________________________    Phosphate Buffered                                                            Saline     (M).sup.b                                                                           99.sup.c                                                                        116  107  158  1.6                                         Pertussis  (M)  113                                                                              108  117  149  1.3                                         PRP (K) (Example 1)                                                                      (M)  110                                                                              136  148  142  1.3                                         PRP (K) (3 mo)                                                                           (S).sup.d                                                                          108                                                                              136  132  147  1.4                                         Pertussis (3mo)                                                                          (M)  101                                                                              135  1002 1771 17.5                                        (Example 5)                                                                   PRP (K)+Pertussis                                                                        (S)   93                                                                              146  135  140  1.5                                         (3 mo) (Example 4)                                                                       (M)  119                                                                              154  751  1410 11.8                                        __________________________________________________________________________     a) 10 μg PRP or 3.5 op units pertussis/dose (0.5 ml).                      b) Booster2nd and 3rd week respectively (multiple injection)                  c) Average 10 rats.                                                           d) Single injection.                                                     

I claim:
 1. A combined vaccine that elicits effective levels of anti-PRP(polyribosyl ribitol phosphate) and anti-pertussis antibody formationsin young warm-blooded animals which consists of polyribosyl ribitolphosphate isolated and purified from the capsular polysaccharide ofHaemophilus influenzae type b by adding hydroxylapatite in about 20millimolar phosphate buffer at pH from about 6.7 to about 6.9, mixing ata temperature of about 1° to 4° C., centrifuging, and removing thesupernatant and repeating the foregoing procedure at least 2 more times,filtering the supernatant, dializing against pyrogen free distilledwater, and then lyophilizing; and Bordetella pertussis antigens.
 2. Acombined vaccine according to claim 1, wherein said Bordetella pertussisantigens are obtained from Bordetella pertussis strain
 138. 3. Acombined vaccine according to claim 1, wherein said polyribosyl ribitolphosphate is obtained from Haemophilus influenzae type b Rab strain. 4.A combined vaccine according to claim 1, wherein said Bordetellapertussis antigens are obtained from Bordetella pertussis strain 138 andsaid polyribosyl ribitol phosphate is obtained from Haemophilusinfluenzae type b Rab strain.
 5. A combined vaccine according to claim1, wherein said young warm-blooded animal is a human child.
 6. Acombined vaccine according to claim 4, wherein said young warm-bloodedanimal is a human child.
 7. A method for inducing active immunizationagainst systemic infection in young warm-blooded animals caused by thepathogen Haemophilus influenzae type b which comprises administering animmunogenic amount of a combined vaccine which consists of polyribosylribitol phosphate, isolated and purified from the capsularpolysaccharide of Haemophilus influenzae type b by addinghydroxylapatite in about 20 millimolar phosphate buffer at pH from about6.7 to about 6.9, mixing at a temperature of about 1° to 4° C.centrifuging, removing the supernatant and repeating the foregoingprocedure at least 2 more times, filtering the supernatant, dializingagainst pyrogen free distilled water, and then lyophilizing; andBordetella pertussis antigens.
 8. A method according to claim 7, whereinsaid systemic infection is meningitis.
 9. A method according to claim 8,wherein said young warm-blooded animal is a human child.